Lehninger 5th Edition 163-166 2l4q64

ti.'l

n a L i g a n d : 0 x y g e n - B iPnrdoitnegi n s n d i nogf a P r o t e ti 0 b li e 5 . 1R e v e r s iB

Lake Powell,Arizona,August 2000.A family was vacationrng in a rented houseboat.They tumed on the electrical generatorto power an air conditionerand a television. About 15 minutes later, two brothers, aged 8 and 11, jumped off the swrmdeck at the stem. Situatedimmediatelybelowthe deckwasthe exhaustport for the generator. Within two minutes,both boys were overcomeby the carbon monoxide in the exhaust,which had becomeconcentrated in the spaceunder the deck.Both drowned.These deaths,alongwith a seriesof deathsin the 1990sthat were linked to houseboatsof similar design,eventuallyled to the recall and redesignof the generatorexhaustassembly. Carbonmonoxide(CO),a colorless,odorlessgas,is responsiblefor more than half of yearly deathsdue to poisoning worldwide. CO has an approximately250-fold greater affinity for hemo$obin than does o4ygen. Consequently, relatively low levels of CO can have substantialand tragic effects.WhenCO combineswith hemo$obin,the complex or COlIb. is referred to as carboxyhemoglobin, produced by natural processes,but loSomeCO is generally result only from human activically high levels ties. Engineand furnaceexhaustsare important sources, as COis abyproduct of the ncomplete combustionof fossil fuels. In the United Statesalone,nearly 4,000people succumbto CO poisoning each year, both accidentally and ntentionally. Many of the accidentaldeathsinvolve undetectedCObuildup in enclosedspaces,suchaswhen a householdfurnace malfunctionsor leaks,venting CO into a home. However,CO poisoningcan also occur in open spaces,as unsuspectingpeopleat work or play inhale the exhaustfrom generators,outboardmotors,tractor engines,recreationalvehicles,or lau,nmowers. Carbonmonoxidelevelsin the atmosphereare rarely dangerous,rangingfrom lessthan 0.05parts per miliion (ppm) in remote and unintrabited areasto 3 to 4 ppm in somecities of the northern hemisphere.In the United States,the government-mandated(OccupationalSafety and Health istration,OSHA)Iimit for CO at worksites is 50 ppm for people working an eight-hour shift. The tight binding of COto hemoglobinmeansthat COHb can accumulate over time as people are exposed to a constantlow-levelsourceof CO. In an average,healthy individual, 1% or less of the total hemoglobinis complexedas COHb.SinceCO is a product of tobacco smoke, many smokershave COHb levelsin the range of 3% to 8% of total hemoglobin,and the levels can rise to l5o/ofor chain-smokers.COHb Ievelsequilibrateat 50% in peoplewho breathe air containing 570 ppm of CO for severalhours. Reliablemethods have been developedthat relate CO content in the atmosphereto COHblevelsin the blood (F€ 1). In tests ofhouseboatswith a generatorexhaustlike the one responsiblefor the Lake Powell deaths,CO levelsreached

6,000to 30,000ppm under the swim deck, and atmospheric 02 levels under the deck declined from 2lo/oto I2o/o.Even above the swim deck, CO levels of up to 7,200ppm were detected, high enoughto causedeath within a few minutes. How is a human affected by COFIb?At levels of less than 10% of total hemo$obin, sSrmptomsare rarely obmild headaches. served.At I5o/o,theindividualexperiences the headacheis severeand is generallyacAt 20o/oto 30o/o, companiedby nausea,dizziness,confusion,disorientation, and somevisual disturbances;these symptomsaxegenerally reversedif the individual is treated with o>rygen.At COIIblevelsof 30%to 5\o/o,theneurologicalsymptomsbecome more severe,and at levels near 50%, the individual and can sink into coma.Respiratory losesconsciousness failure may follow.With prolongedexposure,somedamage becomespermanent.Death normally occurs when COFIb levels rise above 60%0.Autopsy on the boys who died at and\2o/o' LakePowellrevealedCOIIblevelsof 59o/o Binding of CO to hemoglobin is affected by many factors,including exercise(Fig. i) and changesin air pressure related to altitude. Becauseof their higher base levels of COHb,smokersexposedto a sourceof CO often developsymptomsfaster than nonsmokers. Individuals with heart, lung, or blood diseasesthat reduce the availability of oxygen to tissues may also experience symptoms at lower Ievels of CO exposure. Fetuses are at particular risk for CO poisoning,becausefetal hemoglobinhas a somewhathigher affinity for CO than adult hemoglobin.Casesof CO exposure have been recorded in which the fetus died but the mother recovered. (conti'rrued on nent PaSe)

gto 8a E pb

()4

'o 0

20

80 60 40 Carbon monoxide (PPm)

1oo

betweenlevelsof COHb in blood and concenI Relationship FIGURE trationof CO in the surroundingair. Fourdifferentconditionsof exposure are shown, comparing the effects of short versusextended exposure,and exposureat restversusexposureduring light exercise'

i

164

]

ProteF i nu n c t i o n

It may seem surprising that the loss of half of one,s hemoglobin to COHb can prove fatal-we know that people with any of several anemic conditions manage [o function reasonably well with half the usual complement of active hemoglobin. However, the binding of CO to hemoglobin does more than remove protein from the pool available to bind oxygen It also affects the affinity of the remaining hemoglobin subunits for oxygen. As CO binds to one or two subunits of a hemoglobin tetramer, the affinity for 02 is increased substantially in the remaining subunits (Fig 2) Thus, a hemoglobin tetramer wrth two bound CO molecules can efficiently bind 02 in the lungs-but it releases very little of it in the tissues. Oxygen deprivation in the tissues rapidly becomes severe. To add to the problem, the effects of CO are not limited to interference with hemoglobin function. CO binds to other heme proteins and a variety of metalloproteins. The effects of these interactions are not yet well understood, but they may be responsible for some of the longer-term effects of acute but nonfatal CO poisoning. When CO poisoning is suspected, rapid evacuation of the person away from the CO source is essential,but this does not always result in rapid recovery. When an individual is moved from the CO-polluted site to a nor_ mal, outdoor atmosphere, 02 begins to replace the CO in hemoglobin-but the COHb levels drop only slowly. The half-time is 2 to 6 5 hours, depending on individual and environmental factors. If 100% oxygen is istered with a mask, the rate of exchange can be increased about fourfold; the half-time for O2-CO exchange can be reduced to tens of minutes if 100% oxygen at a pressure

The expressionfor 0 (seeEqn 5-8) is o_

[L]" lLl" + Ku

3 (5-14)

Rearranging,then taking the log of both sides, yields

e _ ILl" r-0 Kd h-(*)

:nrogtlJ -rogKd

(5-15)

$ ts-rol

wnereKa: [L]6s. Equation 5-16 is the Hill equation, and a plot of log W/Q 0)l versus log [L] is called a Hill plot. Based on the equation, the Hill plot should have a slope of n However, the experimentally determined slope actually reflects not the number of binding sites but the degree of interaction between them. The slope of a Hill plot is therefore de_ noted by ns, the Hill coefficient, which is a measure of the degree of cooperativity. If nsequals 1, ligand binding is not cooperative, a situation that can arise even in a mul_ tisubunit protein if the subunits do not communicate. An

48I2 pO2(kPa) FIGURE2 Several oxygen-binding curves:for normalhemoglobin, hemoglobinfrom an anemicindividualwith only 50% of her hemoglobin functional,and hemoglobin from an individualwith 50% of his hemoglobinsubunitscomplexedwith CO. The pO2 in humanlungs andtissues is indicated

of 3 atm (303kPa) is supplied.Thus,rapid treatmentby a properly equippedmedicalteam is critical. Carbonmonoxide detectorsin all homesare highly recommended.This is a simple and inexpensivemeasure to avoid possibletragedy.After completingthe research for this box, we immediately purchasedseveral new CO detectorsfor our homes.

n11of greater than 1 indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of others. The theoretical upper limit for ns is reached when ns : ?L In this case the binding would be completely cooperative: all bindiry sites on the protein would bind ligand simultaneously, and no protein mole_ cules partially saturated with ligand wor:ld be present under any conditions. This limit is never reached in practice, and the measured value of ns is always less than the actual number of ligand-binding sites in the protein. Annll of less than I indicates negative cooperativ_ ity, in which the binding of one molecule of ligand i,mpedes the binding of others. Well-documented cases of negative cooperativity are rare. To adapt the Hill equation to the binding of oxygen to hemoglobin we must again substitute pO2 for [L] and P{o for Ka: .-(*)

: nbspoz - nlogp5s

(b-17)

n a L i g a n d : 0 x y g e n - B iPnrdoitnegi n s["t] n d i nogfa P r o t e ti o b li e 5 . 1R e v e r s iB

1 s

Hemoglobin high'a{Iinity state \,, nu: | .u

l*0

l'');",'

-1

Iow-affrnity

-2

_J

-2

-1 log p02

FIGURE 5-14 Hill plots for oxygenbindingto myoglobinand hemo-1, The maximum globin.When nr1: thereis no evidentcooperativity. approxiobservedfor hemoglobincorresponds degreeof cooperativity a highlevelof coopermatelyto ng : 3. Notethatwhilethisindicates sitesin hemoglobin. ativity,ns is lessthann, the numberof O2-binding Thisis normalfor a proteinthat exhibitsallostericbindingbehavior

Hill plots for myoglobin

and hemoglobin

subunitsof a cooperativelybinding protein are functionally identical,that eachsubrnit can exist in (at least) two conformations,and that all subunitsundergothe transition from one conformationto the other simultaneously. In this model, no protein has individual subunits in different conformations. The two conformations are in equilibrium.The hgandcan bind to either conformation, but binds eachwith different affinity.Successivebinding of ligand molecules to the low-affinity conformation (which is more stablein the absenceof ligand) makesa transition to the high-affinity conformationmore likely. In the second model, the sequential model (Fig. 5-15b), proposedin 1966by DanielKoshlandand colleagues,ligandbinding can induce a changeof conformation in an individual subunit. A conformational change in one subunit makes a similar change in an adjacentsubunit, as well as the binding of a secondligand molecule,more likely. There are more potential intermediatestatesin this model than in the concerted model. The two models are not mutually exclusive;the concertedmodelmaybe viewedasthe "all-or-none"limiting caseof the sequentialmodel.In Chapter6 we use thesemodelsto investigateallostericenzyrnes'

H- and(0t Also Transports hlernoglobin

are given in

Figure 5-14.

for TwoModels Suggest Mechanisms Binding [ooperative Biochemistsnow know a great deal about the T and R statesof hemoglobin,but much remainsto be learned about how the T -+ R transition occurs.T\.vomodelsfor the cooperativebinding of ligandsto proteins with multiple binding sites have greatly influenced thinking aboutthis problem. The flrst model was proposedby JacquesMonod, JeffriesW;rman,and Jean-PierreChangeuxin 1965,and is called the MWC model or the concerted model (Fig. 5-15a). The concertedmodel assumesthat the

In addition to carryingnearly all the oxygenrequired by cells from the lungs to the tissues,hemoglobincarries two end products of cellularrespiration-H+ and COzfrom the tissuesto the Iungs and the kidneys,where they are excreted.The CO2,producedby oxidationof organicfuels in mitochondria,is hydrated to form bicarbonate: CO2+ H2Oi-

H* + HCOt

This reaction is catalyzedby carbonic anhydrase, an enzyrneparticularly abundant in erythrocytes. Carbon dioxideis not very solublein aqueoussolution,and bubblesof CO2wouldform in the tissuesandbloodif it were not convertedto bicarbonate.As you can seefrom the

A1lE

AllO

ETI

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1l ul 1l

l=o-E-E-m oo-oo-m-re=1l\ 11 1t 1t lr\tvtv G(])-Im-trt-m-ul

6XD

fl,lLl

GG)-EO-m-m-ffi

r-Y-) XX

1l on (J.,

tTl

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1l

1l trI 1l

6YD

tilL]

aJ

1l

lxlxt

GXD

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(a)

ll 1t

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I

1f 1t 1r 1r\11 oo-oo-er.-[I)=-ul 1l 1t 1t 1t\1t (n{) -nlD -tTlt

-ttTil-EItl

60:60:m-re-ETt

1t 1t 1t 1r\1r

cYD .mD -Etil -tr|il-trit m:EGj=- l-Lft 66:66: ft)

5-15 Two generalmodelsfor the interconFIGURE versionof inactiveand activeforms of a proteinduring cooperativeligandbinding'Althoughthe models may be appliedto any protein-includingany enbinding, 6)-that exhibitscooperative zyme(Chapter we show herefour subunitsbecausethe modelwas originallyproposedfor hemoglobin.(a) In the conmodel(MWC model),all subcerted,or all-or-none, postulated to be in the sameconformation, unitsare (low or inactive)or all tr (high affinity eitherall O K1, on the equilibrium, affinityor active).Depending betweenO and D forms,the bindingof one or more (L)will pull the equilibriumtoward ligandmolecules with boundL areshaded.(b) ln the ! form.Subunits model,eachindividualsubunitcan be the sequential in eitherthe O or n form.A very largenumberol is thusPossible. conformations

!"1 P r o t e Fi nu n c t i o n reaction catalyzedbycarbonicanhydrase,the hydration of CO2resultsin an increasein the H+ concentration(a decreasein pH) in the tissues.The bindingof oxygenby hemoglobinis profoundly influenced by pH and CO2 concentration,so the interconversionof CO2and bicarbonate is of great importance to the regulation of oxygenbinding and releasein the blood. Hemoglobintransports abottt 400/o of the total H+ and 15% to 20o/oof the CO2formed in the tissues [o the lungs and kidneys.(The remainderof the H+ is absorbedby the plasma'sbicarbonatebuffer; the remainder of the CO2is transportedas dissolvedHCO| and CO2.)The binding of H* and CO2is inverselyrelated to the binding of oxygen.At the relativelyIow pH and high CO2 concentrationof peripheral tissues, the affinity of hemoglobinfor oxygendecreasesas H+ and CO2are bound,and 02 is releasedto the tissues.Conversely, in the capillaries of the lung, as CO2 is excreted and the blood pH consequentlyrises, the affinity of hemoglobinfor oxygen increasesand the protein binds more 02 for transport to the peripheral tissues.This effect of pH and CO2 concentrationon the binding and release of oxygen by hemoglobinis calledthe Bohr effect, after ChristianBohr, the Danish physiologist (and father of physicist Niels Bohr) who discoveredit in 1904. The binding equilibrium for hemoglobinand one molecule of oxygen can be designatedby the reaction Hb + 02 -+

HbO2

but this is not a completestatement.To for the effect of H+ concentrationon this binding equilibrium, we rewrite the reaction as HHb*+Oz#HbOr+H+ where HlIb+ denotesa protonatedform of hemoglobin. This equationtells us that the O2-saturationcurveof hemo$obin is influencedby the H* concentration(Fig. b-f 6). Both 02 and H* are boundby hemoglobin,but with inverse afnniff. When the oxygen concentrationis high, as in the lungs, hemo$obin binds 02 and releasesprotons.When the oxygen concentrationis low; as in the peripheral tissues,H* is boundand 02 is released. Oxygenand H+ are not bound at the samesites in hemoglobin.Oxygen binds to the iron atoms of the hemes,whereasH* binds to any of severalamino acid residuesin the protein. A major contribution to the Bohr effect is made by Hisra6(His HC3) of the B subunits. When protonated,this residueforms one of the ion pairs-to Aspea(Asp FGI)-that helps stabilize deoxyhemoglobinin the T state (Fig. 5-9). The ion pair stabilizesthe protonated form of His HCB,giving this residue an abnormally high pK, in the T state. The pK. falls to its normal value of 6.0 in the R state becausethe ion pair cannot form, and this residue is largely unprotonatedin oxyhemoglobinat pH 2.6, the blood pH in the lungs. As the concentrationof H+

o o.s

0246810 pO2 ftPa) FIGURE 5-16 Effectof pH on oxygenbindingto hemoglobin.ThepH o f b l o o di s 7 . 6 i n t h e l u n g sa n d 7 . 2i n t h e t i s s u e E s .x p e r i m e n tmael a surements on hemoglobinbindingare oftenperformedat pH 7.4.

rises, protonation of His HC3 promotes release of oxygen by favoring a transition to the T state. protonation of the amino-terminalresidues of the a subunits, certain other His residues,and perhaps other groups has a similar effect. Thus we seethat the four polypeptide chainsof hemoglobin communicatewith each other not only about 02 binding to their hemegroupsbut alsoabout H+ brnding to specificamino acid residues.And there is still more to the story.Hemoglobinalsobinds CO2,againin a manner inverselyrelated to the binding of oxygen.Carbon dioxide binds as a carbamategroup to the a-amino group at the amino-terminalend of each globin chain, forming carbaminohemoglobin:

o C+

o

TT-

H2N-C-C-

--l--)

RO

Amino-terminal residue

Oi TT

C-N-C-C-

ltttl o Ro Carbamino-terminal residue

This reaction produces H+, contributing to the Bohr effect. The bound carbamates also form additional salt bridges (not shown in Frg 5-9) that help to stabilize the T state and promote the release of oxygen. When the concentration of carbon dioxide is high, as in peripheral tissues, some CO2 binds to hemoglobin and the affinity for 02 decreases, causing its release. Conversely, when hemoglobin reaches the lungs, the high oxygen concentration promotes binding of 02 and release of CO2. It is the capacity to communicate ligandbinding information from one potypeptide subunit to the others that makes the hemoglobin molecule so beautifully adapted to integrating the transport of 02, CO2, .--+. and .t-l by en'throcytes.